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1.
Nat Immunol ; 23(5): 692-704, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35484407

RESUMO

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Trifosfato de Adenosina/metabolismo , Transporte de Elétrons , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Mol Cell ; 83(6): 1012-1012.e1, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36931250

RESUMO

Mitochondria have emerged as signaling organelles with roles beyond their well-established function in generating ATP and metabolites for macromolecule synthesis. Healthy mitochondria integrate various physiologic inputs and communicate signals that control cell function or fate as well as adaptation to stress. Dysregulation of these mitochondrial signaling networks are linked to pathology. Here we outline a few modes of signaling between the mitochondrion and the cytoplasm. To view this SnapShot, open or download the PDF.


Assuntos
Mitocôndrias , Transdução de Sinais , Mitocôndrias/metabolismo , Citoplasma/metabolismo , Organelas/metabolismo , Aclimatação
3.
Nature ; 602(7896): 321-327, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34937051

RESUMO

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.


Assuntos
COVID-19/sangue , COVID-19/imunologia , Células Dendríticas/imunologia , Interferons/imunologia , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Brônquios/imunologia , Brônquios/virologia , COVID-19/patologia , Chicago , Estudos de Coortes , Progressão da Doença , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Imunidade Inata , Londres , Masculino , Mucosa Nasal/imunologia , Mucosa Nasal/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Traqueia/virologia , Adulto Jovem
4.
Nature ; 615(7952): 401-402, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36890308
5.
Proc Natl Acad Sci U S A ; 113(27): E3844-51, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27335462

RESUMO

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.


Assuntos
Vírus da Parainfluenza 5/metabolismo , Proteínas Virais de Fusão/metabolismo , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Conformação Molecular , Mutação , Estabilidade Proteica , Células Vero
8.
Proc Natl Acad Sci U S A ; 111(25): E2596-605, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24927585

RESUMO

To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.


Assuntos
Vírus da Parainfluenza 5/química , Peptídeos/química , Redobramento de Proteína , Proteínas Virais de Fusão/química , Oxirredução
9.
J Virol ; 89(6): 3438-41, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25589638

RESUMO

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.


Assuntos
Vírus da Parainfluenza 5/metabolismo , Proteínas Virais de Fusão/química , Motivos de Aminoácidos , Cristalografia por Raios X , Humanos , Mutação , Vírus da Parainfluenza 5/química , Vírus da Parainfluenza 5/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Infecções por Rubulavirus/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
10.
Bioconjug Chem ; 25(4): 707-17, 2014 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-24670265

RESUMO

Post-assembly functionalization of supramolecular nanostructures has the potential to expand the range of their applications. We report here the use of the chemoselective native chemical ligation (NCL) reaction to functionalize self-assembled peptide amphiphile (PA) nanofibers. This strategy can be used to incorporate specific bioactivity on the nanofibers, and as a model, we demonstrate functionalization with the RGDS peptide following self-assembly. Incorporation of bioactivity is verified by the observation of characteristic changes in fibroblast morphology following NCL-mediated attachment of the signal to PA nanofibers. The NCL reaction does not alter the PA nanofiber morphology, and biotinylated RGDS peptide was found to be accessible on the nanofiber surface after ligation for binding with streptavidin-conjugated gold nanoparticles. In order to show that this strategy is not limited to short peptides, we utilized NCL to conjugate yellow fluorescent protein and/or cyan fluorescent protein to self-assembled PA nanofibers. Förster resonance energy transfer and fluorescence anisotropy measurements are consistent with the immobilization of the protein on the PA nanofibers. The change in electrophoretic mobility of the protein upon conjugation with PA molecules confirmed the formation of a covalent linkage. NCL-mediated attachment of bioactive peptides and proteins to self-assembled PA nanofibers allows the independent control of self-assembly and bioactivity while retaining the biodegradable peptide structure of the PA molecule and thus can be useful in tailoring design of biomaterials.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Nanoestruturas/química , Peptídeos/química , Peptídeos/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Fibroblastos/citologia , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Tamanho da Partícula , Propriedades de Superfície
11.
Nat Metab ; 6(8): 1492-1504, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39048801

RESUMO

Microglia are necessary for central nervous system (CNS) function during development and play roles in ageing, Alzheimer's disease and the response to demyelinating injury1-5. The mitochondrial respiratory chain (RC) is necessary for conventional T cell proliferation6 and macrophage-dependent immune responses7-10. However, whether mitochondrial RC is essential for microglia proliferation or function is not known. We conditionally deleted the mitochondrial complex III subunit Uqcrfs1 (Rieske iron-sulfur polypeptide 1) in the microglia of adult mice to assess the requirement of microglial RC for survival, proliferation and adult CNS function in vivo. Notably, mitochondrial RC function was not required for survival or proliferation of microglia in vivo. RNA sequencing analysis showed that loss of RC function in microglia caused changes in gene expression distinct from aged or disease-associated microglia. Microglia-specific loss of mitochondrial RC function is not sufficient to induce cognitive decline. Amyloid-ß plaque coverage decreased and microglial interaction with amyloid-ß plaques increased in the hippocampus of 5xFAD mice with mitochondrial RC-deficient microglia. Microglia-specific loss of mitochondrial RC function did impair remyelination following an acute, reversible demyelinating event. Thus, mitochondrial respiration in microglia is dispensable for proliferation but is essential to maintain a proper response to CNS demyelinating injury.


Assuntos
Proliferação de Células , Doenças Desmielinizantes , Microglia , Mitocôndrias , Animais , Microglia/metabolismo , Camundongos , Mitocôndrias/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Respiração Celular
12.
JCI Insight ; 9(8)2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38502186

RESUMO

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.


Assuntos
Citocinas , Pulmão , Microglia , Pneumonia , Citocinas/metabolismo , Pulmão/metabolismo , COVID-19 , Encéfalo , Autopsia , Humanos , Camundongos , Disfunção Cognitiva , Imunofluorescência , Pneumonia/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
bioRxiv ; 2023 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-37546860

RESUMO

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-α and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2-molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

14.
Cytoskeleton (Hoboken) ; 74(9): 317-330, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28646493

RESUMO

Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.


Assuntos
Cinesinas/metabolismo , Mutação de Sentido Incorreto , Fuso Acromático/metabolismo , Quinases da Família src/metabolismo , Substituição de Aminoácidos , Humanos , Cinesinas/química , Cinesinas/genética , Fosforilação , Fuso Acromático/química , Fuso Acromático/genética , Quinases da Família src/química , Quinases da Família src/genética
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